Use of oyster flesh enzymatic hydrolysates for preparing compositions eliminating free radicals

ABSTRACT

The invention concerns the use of an oyster enzymatic hydrolysate for preparing a composition eliminating free radicals. The invention is characterized in that said hydrolysate is obtainable by hydrolysis of oyster flesh by a protease. The invention is applicable in therapy, dietetics and cosmetology.

The present invention relates to the use of enzymatic oyster fleshhydrolysates for preparing free-radical scavenging compositions, of usein particular in therapeutics, in dietetics and in cosmetology.

Oxygenated free radicals are atoms or molecules which have an unpairedelectron in their outer orbital (OH—, O₂ ⁻, ROO⁻, RO⁻, etc.). As aresult, they are extremely unstable and can react with stable moleculessuch as lipids, carbohydrates, proteins or nucleic acids, which arefundamental elements of cells, so as to pair their electron, thisreaction leading to a chain reaction of formation of new free radicals.Thus, they are capable of causing serious cellular modifications, suchas mutation or cellular ageing, or even cell death.

At the cellular level, oxygenated free radicals are constantly beingformed. They may also form during detoxification mechanisms afterexposure to certain substances or under the effect of radiation.Normally, the endogenous production of oxygenated free radicals iscounterbalanced by the presence of defense systems represented, firstly,by enzymes (superoxide dismutases, catalases, glutathione peroxidases)which intercept the active forms of the oxygen and, secondly, by “agentswhich trap free radicals” (glutathione, uric acid, vitamin C, vitamin A,vitamin E, taurine, etc.) which block membrane lipid peroxidation chainreactions, such that the organisms are not harmed by them.

However, many situations may lead to the excessive formation ofoxygenated free radicals: intense exposure to sunlight, intoxicationwith certain chemical products and certain medicinal products, abruptreoxygenation or hyperoxygenation of tissues previously deprived ofoxygen, the occurrence of an intense (burns, infections, etc.) orchronic inflammatory reaction. An excess of oxygenated free radicals mayalso be linked to a genetic disease or to a decrease in defenses:immaturity of enzymatic systems in newborn babies, ageing, dietarydeficiencies in vitamins and in trace elements (selenium, zinc, etc.).

Be that as it may, some responsibility in the genesis and maintenance ofa certain number of chronic pathologies, such as atherosclerosis, malignant conditions, inflammatory pathologies (Crohn's disease for example)and neurodegenerative pathologies (Alzheimer's disease, Parkinson'sdisease, etc.), or ageing, and also acute pathologies, such aspost-ischemic reperfusion lesions, burns, septic shocks, viralinfections, serious infectious conditions and allergies, have beenattributed to an imbalance between the production and destruction ofoxygenated free radicals, without it always being possible to specifywhether these free radicals are the cause or the consequence, or bothsimultaneously, of the disease.

Consequently, it is understandable that a very large number of studiesshould currently be directed toward understanding more clearly theinvolvement of oxygenated free radicals in physiopathology, and towarddeveloping compounds or compositions able to oppose the deleteriouseffects of these free radicals.

Some authors (Livingstone et al., 1990, Funct. Ecol. 4, 415-424; Regoliand Principato, 1995, Aquat. Tox., 31, 143-164) have demonstrated, inmarine molluscs, not only the presence of superoxide dismutases, ofcatalases and of glutathione peroxidases, but also that of specificantioxidant enzymes such as glyoxalase, which catalyses thedetoxification of ketoaldehydes formed during oxidative stress, andglutathione transferases, which catalyse a large variety of reactionsconjugating glutathione to xenobiotic compounds, indicating that theseorganisms are capable of protecting themselves against oxygenated freeradicals. Moreover, antioxidants, such as glutathione, vitamin A,vitamin E and taurine, have been detected in marine molluscs and haveproved, in certain cases, to be quantitatively proportional to theoxidative stress experienced by these animals.

Thus, it has become apparent that marine molluscs may constitute asource of free-radical scavenging compounds which can be used in theprevention and treatment of the harmful effects of oxygenated freeradicals.

A certain number of authors have more especially taken an interest inthe free-radical scavenging potentials of oyster extracts. Inparticular:

-   -   Tapiero and Tew (Biomed. & Pharmacother., 1996, 50, 149-153)        have studied the effects of an oyster lyophilisate, named JCOE        (Japan Clinic Oyster Extract) on the intracellular content of        glutathione-stimulating hormone (GSH), and also on the activity        of glutathione-S-transferase (GST), of a culture of HL60 cells.        This lyophilisate is obtained by heating oyster flesh at 80° C.        for 1 hour then subjecting the resulting product to        centrifugation and lyophilizing the supernatant thus collected.        Tapiero and Tew thus demonstrated a significant increase in GSH        synthesis in the HL60 cells cultured in the presence of the        lyophilisate, without, however, noting any significant        modification of the activity of GST.    -   Yoshikawa et al. (Biomed. & Pharmacother., 1997, 51, 328-332)        have shown that an oyster lyophilisate, JCOE, is capable, in        vitro, of trapping superoxide and hydroxyl radicals and of        protecting rat gastric mucosa cells against the deleterious        effects of hydrogen peroxide, when these cells are pretreated        for 24 hours with this lyophilisate.    -   Kimura et al. (Journal of Ethnopharmacology, 1998, 59, 117-123)        have shown that rats which are fed on peroxidized corn oil and        receive, twice a day and orally, an aqueous oyster extract have        serum levels of free fatty acids, triglycerides and lipid        peroxides, and a hepatic level of total cholesterol, which are        lower than those observed in rats fed in the same way but which        do not receive aqueous oyster extract. Moreover, these authors        have demonstrated the presence, in this aqueous extract, of a        substance capable of both inhibiting adrenaline-induced        lipolysis and stimulating lipogenesis from glucose in rat fat        cells, which they have identified as being adenosine.    -   Nomura et al. have proposed, in the European Patent Application        published under the no. 0 806 465 in the name of Japan Clinic        Co. Ltd., preparing an antioxidant composition using a method        consisting in fractionating with ethanol an aqueous oyster        extract obtained beforehand by heating a mixture of oyster flesh        and water at a temperature of between 50 and 90° C. for 2 to 3        hours. The antioxidant properties of the composition thus        prepared are demonstrated, in that patent application, via tests        aimed at assessing its ability to inhibit, in vitro, the        reaction between superoxide anions produced by an enzymatic        system xanthine—xanthine oxydase and        5,5-dimethyl-1-pyrrolen-1-oxide.    -   Dussart (Ifremer Report: Stage de VIème Année [6th Year training        period], 1997, Faculté de Pharmacie, [School of Pharmacy],        Université de Lille II [Lille II University]) has carried out a        study aimed at comparing the free-radical scavenging properties        of aqueous oyster extracts prepared by mixing material from        ground oyster flesh with dionized water and then subjecting the        resulting mixture to centrifugation followed by lyophilization        of the supernatant, with those exhibited by oyster extracts        prepared by subjecting material from grinding oysters to        lyophilization only. The results of this study show that, while        both types of oyster extract have, in vitro, a protective effect        against oxidations induced, firstly, by a generator of peroxide        radicals on hematocytes and, secondly, by copper on low density        lipoproteins (LDLs), the aqueous oyster extracts appear to        exhibit the most advantageous antioxidant potential.

It has, moreover, been proposed, in the Japanese Patent Applicationspublished under no. 7-082132 and no. 7-102252, to use, in cosmeticcompositions, hydrolysates prepared from oyster mucus, as antioxidantagents able to prevent skin ageing and in particular the appearance ofwrinkles. These hydrolysates are obtained by centrifuging or pressingoysters, after extraction from their shells, and removing the flesh.

The mucus is then subjected to a series of fractionations with ethanolto rid it of the sodium chloride which it contains, and then toproteolysis. In Japanese Patent Application No. 7-102252, the mucus,once hydrolyzed, is subjected to a further desalifying operation, againusing ethanol, in order to decrease its coloration.

The cost of manufacturing such hydrolysates is very high, in particularbecause of the not insignificant amounts of ethanol used during thedesalifying operations and the need to have specific and relativelyexpensive installations due to the use of organic solvents. Because ofthis, regardless of knowing whether they exhibit significantfree-radical scavenging activity, it is not desirable to use this typeof hydrolysate for manufacturing free-radical scavenging compositions onan industrial scale, in particular if these compositions are intended tobe marketed as food supplements.

Now, in the context of their studies, the inventors have noted thatoyster hydrolysates obtained by subjecting oyster flesh to the action ofa protease under suitable conditions exhibit, surprisingly, afree-radical scavenging activity which is even higher than that observedfor the aqueous oyster extracts tested by Dussart in the abovementionedstudy, and may therefore advantageously be used for manufacturingcompositions intended to prevent or treat the deleterious effects ofoxygenated free radicals.

A subject of the present invention is therefore the use of an enzymaticoyster hydrolysate for preparing a free-radical scavenging composition,this use being characterized in that said hydrolysate can be obtained byhydrolyzing oyster flesh using a protease.

According to a first advantageous arrangement of the invention, thehydrolysis of the oyster flesh is carried out using a protease chosenfrom subtilisin, pepsin and trypsin. In fact, besides the fact that thecost of these proteases is compatible with industrial exploitation ofthe invention, they have the advantage of being part of the enzymeswhose use is authorized in a large number of countries for preparingprotein hydrolysates used in manufacturing food supplements.

Since proteases are not all active within the same pH and temperatureranges, the pH and temperature conditions under which the hydrolysis ofthe oyster flesh is performed depend on the protease chosen to carry outthis hydrolysis.

Preferably, these pH and temperature conditions are such that they makeit possible to obtain optimum activity of the protease. Thus, forexample, the hydrolysis is preferentially carried out at a pH ofapproximately 8 and a temperature of approximately 60° C. in the case ofsubtilisin, at a pH of approximately 2 and a temperature ofapproximately 40° C. in the case of pepsin, and at a pH of approximately8 and a temperature of approximately 37° C. in the case of trypsin.

According to another advantageous arrangement of the invention, thehydrolysis of the oyster flesh is carried out for a period of timesufficient for the hydrolysate to exhibit a degree of protein hydrolysisat least equal to 30%, and preferably to 50%, this degree of proteinhydrolysis being determined by the equation below (Adler-Nissen, 1977,Proc. Biochem., 12, 18-23):DH=(h/h total)×100in which:

-   -   h total represents the total number of peptide bonds present in        the oyster flesh at the start of hydrolysis, whereas    -   h represents the number of peptide bonds hydrolyzed during the        hydrolysis, and is determined by the difference between the        number of free amino (or carboxylic) ends present in the        hydrolysate at the end of the hydrolysis (h₁) and the number of        free amino (or carboxylic) ends present in the ground material        at the start of the hydrolysis (h₀).

For the purposes of the present invention, the start of the hydrolysiscorresponds to the moment at which the protease is brought into contactwith the oyster flesh, while its end corresponds to the moment at whichthe hydrolysis is stopped by inactivation of said protease, for exampleby heat denaturation or by modification of the pH.

The total number of peptide bonds (h total) present in the oyster fleshcan be obtained by the difference between the amount of total aminoacids (free+bound) and the amount of free amino acids which this fleshcontains. These amounts of total and free amino acids may be determined,for example, using a kit such as that marketed under the trademarkWaters AccQ-Tag Chemistry Package® by the company Waters. The number ofpeptide bonds hydrolyzed (h) during the hydrolysis is, itself, obtainedby the difference between the amount of free amino ends (h₁) present inthe hydrolysate at the end of the hydrolysis and the amount of freeamino ends (h₀) present in the oyster flesh at the start of thehydrolysis, which can be determined, for example, by reaction withfluorodinitrobenzene according to the protocol described in Biochem. J.,45, 563, 1949.

Here also, the amount of time for which the hydrolysis should be allowedto proceed in order to obtain a hydrolysate having a degree of proteinhydrolysis at least equal to 30%, and preferably to 50%, depends on theprotease chosen to carry out this hydrolysis and, for the same protease,on the pH and temperature conditions under which the hydrolysis iscarried out and also on the dose at which this protease is used, thehydrolysis in fact occurring more rapidly, the higher the dose ofprotease.

According to another advantageous arrangement of the invention, thehydrolysate may be obtained using a method comprising, prior to thehydrolysis, an operation consisting in draining the oyster flesh. Inaccordance with the invention, this operation may be carried out bysimply leaving the oysters, once extracted from their shells, to standin a drainer, preferably at a temperature of between 4 and 8° C. so asto prevent any modification of the flesh, this being until no moreliquid flows into said drainer.

According to a preferred arrangement of the invention, the hydrolysatemay be obtained using a method comprising, prior to the hydrolysis, anoperation consisting in grinding the oyster flesh, optionally followedby an operation consisting in diluting the resulting ground material inwater.

Particularly preferably, the grinding operation is carried out after anoperation consisting in draining the oyster flesh.

According to yet another advantageous arrangement of the invention, thehydrolysis is stopped by heat denaturation of the protease.

According to yet another advantageous arrangement of the invention, thehydrolysate may be obtained using a method which also comprises anoperation consisting in collecting the liquid phase of the hydrolysateas it is at the end of the hydrolysis. This collection, which isintended to eliminate the various debris (shell debris, membrane debris,etc.) which may be present in this hydrolysate, may be carried out usingany techniques conventionally used to separate a liquid phase from asolid phase, such as centrifugation, ultra-centrifugation, filtration ormicrofiltration, these techniques possibly being advantageously combinedwith one another.

According to another preferred arrangement of the invention, thehydrolysate may be obtained using a method which comprises the followingsteps:

-   -   a) grinding predrained oyster flesh,    -   b) diluting the ground material in water, at a ground        material/water ratio of between 30/70 and 70/30 (m/v), and        preferably between 40/60 and 60/40 (m/v),    -   c) hydrolyzing the ground material thus diluted with subtilisin        at a pH of approximately 8 and at a temperature of approximately        60° C. for a period of time sufficient for the hydrolysate to        exhibit a degree of protein hydrolysis at least equal to 50%,    -   d) stopping the hydrolysis by inactivation of the subtilisin,        and    -   e) collecting the liquid phase of the hydrolysate.

According to yet another preferred arrangement of the invention, thehydrolysate may be obtained using a method which comprises the followingsteps:

-   -   a) grinding predrained oyster flesh,    -   b) diluting the ground material in water, at a ground        material/water ratio of between 30/70 and 70/30 (m/v), and        preferably between 40/60 and 60/40 (m/v),    -   c) hydrolyzing the ground material thus diluted with pepsin, at        a pH of approximately 2 and at a temperature of approximately        400° C., for a period of time sufficient for the hydrolysate to        exhibit a degree of protein hydrolysis at least equal to 50%,    -   d) stopping the hydrolysis by inactivation of the pepsin, and    -   e) collecting the liquid phase of the hydrolysate.

According to yet another preferred arrangement of the invention, thehydrolysate may be obtained using a method which comprises the followingsteps:

-   -   a) grinding predrained oyster flesh,    -   b) diluting the ground material in water, at a ground        material/water ratio of between 30/70 and 70/30 (m/w), and        preferably between 40/60 and 60/40 (m/v),    -   c) hydrolyzing the ground material thus diluted with trypsin, at        a pH of approximately 8 and at a temperature of approximately        37° C., for a period of time sufficient for the hydrolysate to        exhibit a degree of protein hydrolysis at least equal to 50%,    -   d) stopping the hydrolysis by inactivation of the trypsin, and    -   e) collecting the liquid phase of the hydrolysate.

Such enzymatic oyster hydrolysates have free-radical scavengingproperties which, besides being pronounced, are extremely advantageoussince they prove to be capable not only of neutralizing the effects ofoxygenated free radicals produced during peroxidation reactions, butalso of preventing the formation of these free radicals, through whatappears to be a mechanism of chelation of the metals, such as forexample copper, which are involved in the genesis of said free radicals.

In addition, they have the advantage of being possible to obtain using amethod which is simple to carry out and economically compatible withindustrial demands, in particular due to the fact that it requires theuse of no organic solvent.

These hydrolysates are therefore capable of advantageously being usedfor preparing:

-   -   pharmaceutical compositions intended to treat pathologies which        appear to be linked to an imbalance between the production and        the destruction of oxygenated free radicals, as mentioned above,    -   food supplements suitable for use either as adjuvants to a        medical treatment or in a preventive capacity, in particular by        individuals in whom it is desirable to reinforce the natural        mechanisms of defense against oxygenated free radicals, because        these defense means are physiologically decreased (elderly        individuals, individuals suffering from dietary deficiencies in        vitamins and trace elements, etc.) or because these individuals        are led to find themselves in situations which promote the        excessive formation of oxygenated free radicals (intense        exposure to sunlight, exposure to chemical products, etc.), or    -   cosmetic compositions aimed at preventing or treating skin        ageing, the cause of which is largely inked to the free radicals        generated in the skin by ultraviolet radiation.

To this end, they may be used either as they are, i.e. in aqueous formor, optionally, in the form of dry powders obtained, for example, bylyophilization, or mixed with physiologically acceptable excipientsand/or other active substances, and in particular substances also havingintrinsic free-radical scavenging properties and capable of actingsynergistically (vitamins A, C or E, for example), within more complexformulations.

Besides the arrangements above, the invention also comprises otherarrangements which will emerge from the further description whichfollows, which refers to examples illustrating the hydrolytic activityof enzymes on ground materials from oyster flesh, the preparation ofenzymatic oyster flesh hydrolysates and also the biological propertiesof these hydrolysates, and which refers to the attached drawings inwhich:

FIG. 1 represents the kinetics of two hydrolyses carried out on groundmaterials from oyster flesh, with two different doses of subtilisin;while

FIG. 2 represents the kinetics of a hydrolysis carried out on groundmaterial from oyster flesh with pepsin.

It goes without saying, however, that these examples are given only byway of illustration of the subject of the invention, and in no wayconstitute a limitation thereof.

EXAMPLE 1 Study of the Hydrolytic Activity of Subtilisin on GroundMaterials from Oyster Flesh

Live Crassostrea gigas hollow oysters, originating from the Ifremerexperimental shellfish breeding station at Bouin (Vendée—France), afterextraction from their shells, are drained on a metal sieve for 1 hour ata temperature of between 4 and 8° C., and then ground for 2 minutes at1000 rpm using an Ultra-Turrax® (maximum power equal to 170 W at 2000rpm).

The ground material obtained, after optional storage at a temperature of−20° C. and, in that case, thawing, is introduced into a reactor. 60%(v/m) of deionized water are added, with stirring. A dose of 14 AU(active units) or of 38 AU of subtilisin (marketed under the trademarkalcalase® 2.4 L by the company Novo Nordisk) per kg of the mixture whichthe reactor contains are then introduced into it, still with stirring.The temperature of the reactor is maintained at 60° C. throughout thehydrolysis, i.e. for 4 hours. The stirring is also maintained and the pHis regulated using a pH-stat so as to be constantly at a value of 8.

After 4 hours of hydrolysis, the subtilisin activity is stopped by heatdenaturation of the latter, by placing the reaction mixture in a washbath at 90° C. for 25 minutes.

Samples are taken from the reactor, by means of a peristaltic pump, justbefore the subtilisin is introduced therein (t₀), then 15 and 30 minutesafter the introduction of this enzyme into the reactor (i.e. at t₁₅ andt₃₀), and then every 30 minutes, this being until the hydrolysis isstopped (i.e. at t₆₀, t₉₀, t₁₂₀, t₁₅₀, t₁₈₀, t₂₁₀ and t₂₄₀). The sampleswhich contain the subtilisin are placed in a water bath at 90° C. for 25minutes so as to stop the activity of the latter. All the samples arethen subjected to centrifugation at 13,000 rpm. The supernatants arefiltered through a 0.7 μm membrane, and then through a 0.16 μm membrane.

The hydrolytic activity of the subtilisin is assessed by monitoring:

-   -   firstly, the evolution of the concentration in the ground        materials of free amino ends between t₁₅ and t₂₄₀, by assaying        these ends by reaction with fluorodinitrobenzene, this        monitoring making it possible to establish the kinetics of the        hydrolysis, and    -   secondly, the evolution of the degree of protein hydrolysis (DH)        of the ground materials between t₁₅ and t₂₄₀, this degree of        protein hydrolysis being calculated according to the equation        (DH=(h/h total)×100, in which h total is obtained by assaying        the total and free amino acids present in the ground materials        using a Waters AccQ-Tag Chemistry Package® kit, while h is        determined by assaying the free amino ends present in the        samples taken at t₁₅, t₃₀, etc., up to t₂₄₀ inclusive, by        reaction with fluorodinitrobenzene.

FIG. 1 represents the kinetics of the hydrolysis carried out with the 14AU/kg (▪) dose of subtilisin and that carried out with the 38 AU/kg (♦)dose of subtilisin, the values of the concentrations of free amino endsbeing expressed in mM along the y-axis and the time being expressed inminutes along the x-axis.

This figure shows that the hydrolysis is more rapid when the subtilisindose is increased. Thus, the plateau is reached after 90 minutes ofhydrolysis for the 14 AU/kg dose, and this period of time is reduced to60 minutes for 38 AU/kg dose. However, the concentration of free aminoends for which the plateau is reached is similar for both doses ofenzyme. The same is true for the final concentration of free amino ends(approximately 120 mM).

Table I below shows the values of the degrees of protein hydrolysis(DH), expressed as percentages, obtained for each of the subtilisindoses.

TABLE I Time DH (%) (minutes) 14 AU/kg 38 AU/kg 15 14 — 30 23 31 60 3446 90 45 48 120 47 51 150 50 51 180 47 56 210 54 54 240 54 58

This table shows that, whatever the subtilisin dose used, the rate ofhydrolysis decreases when 45% of the potentially hydrolyzable peptidebonds have been broken. The hydrolysis continues, however, but lightly,since the final values of the degree of protein hydrolysis exceed 50%,to reach 54% in one case and 58% in the other case.

EXAMPLE 2 Study of the Hydrolytic Activity of Pepsin on Ground Materialsfrom Oyster Flesh

The hydrolytic activity of pepsin on ground materials from oyster fleshis assessed using a procedure identical to that used in example 1, withthe exception that the hydrolysis is carried out with a dose of 1% bymass of pepsin relative to the total mass of the groundmaterial/deionized water mixture present in the reactor, at atemperature of 40° C. and at a pH equal to 2.

FIG. 2 represents the kinetics of the hydrolysis thus obtained, thevalues of the concentrations of free amine functions being expressed inmM along the y-axis, the time being expressed in minutes along thex-axis.

This figure shows that the hydrolysis clearly takes place more rapidlythan when it is carried out with subtilisin, even at the dose of 38AU/kg, since the plateau is reached 30 minutes after introducing thepepsin into the reactor. However, the final concentration of free aminefunctions in the hydrolysate, which is around 120 mM, is entirelycomparable to that obtained when hydrolysis is carried out withsubtilisin.

EXAMPLE 3 Preparation of Enzymatic Oyster Flesh Hydrolysates UsingSubtilisin

On the basis of the results obtained in the study which is the subjectof example 1, two hydrolysates which exhibit different degrees ofprotein hydrolysis are prepared—which will hereinafter be named,respectively, hydrolysate A and hydrolysate B—by subjecting two groundmaterials from predrained oyster flesh to hydrolysis with subtilisin.

The ground materials from oyster flesh are prepared and the hydrolysesare carried out under the same conditions as those described in example1, using a subtilisin dose of 38 AU per kg of ground material/deionizedwater mixture.

For hydrolysate A, the hydrolysis is stopped 4 hours after introductionof the enzyme into the reactor, so that it exhibits a maximum degree ofprotein hydrolysis, i.e. close to 60%.

For hydrolysate B, the hydrolysis is stopped 30 minutes afterintroduction of the enzyme into the reactor, so that it exhibits adegree of protein hydrolysis substantially equal to half the maximumdegree of protein hydrolysis, i.e. approximately 30%.

In both cases, the hydrolytic activity of the subtilisin is stopped byplacing the reaction mixtures in a water bath at 90° C. for 25 minutes.The mixtures are then centrifuged at 4000 rpm. The supernatants arefiltered through a 0.7 μm membrane and then through a 0.16 μm membrane.The hydrolysates thus prepared have a granular appearance browny-greenin color. They are lyophilized and placed in flasks at −20° C.

EXAMPLE 4 Biochemical characterization of an Enzymatic Oyster FleshHydrolysate Obtained in Accordance with the Invention

A study is carried out aimed at determining, for hydrolysate A preparedaccording to example 3:

-   -   its solids content,    -   its content of inorganic material,    -   its content of soluble proteins,    -   its content of total sugars and of glycogen, and also    -   its content and its composition of total amino acids and of free        amino acids,        and at comparing the results with those obtained under the same        conditions, firstly, for ground material from oyster flesh        prepared as described in example 1 and, secondly, for an aqueous        oyster extract prepared:    -   by mixing ground material from oyster flesh with deionized water        (1/3, v/v) until a homogeneous mixture is obtained, then    -   subjecting the resulting mixture to centrifugation at 3000 g for        20 minutes, and    -   lyophilizing the supernatant collected at the end of this        centrifugation.

The solids content is determined by placing samples of hydrolysate A ata temperature of 100° C. until a constant weight is obtained (6 hoursminimum) and calculating the percentage represented by this weightrelative to the initial weight of these samples.

The content of inorganic material is determined by incinerating samplesof hydrolysate A at a temperature of 600° C. for 12 hours andcalculating the percentage represented by the weight of the residuerelative to the weight of the solids.

The soluble proteins are assayed using the kit marketed by the companyPierce under the commercial name BCA® Protein Assay Reagent. Bovinealbumin is used as a standard.

The total sugars and the glycogen are assayed according to the methoddescribed by M. Dubois et al., (Anal.

Chem., 1956, 28, 350-356). For these assays, the samples are delipidizedbeforehand according to the method of E. G. Blight and W. J. Dyer (Can.J. Biochem. Physiol., 1959, 37, 911-917).

The content and the composition of total amino acids and of free aminoacids are, themselves, determined using a Waters AccQ-Tag ChemistryPackage® kit. For assaying the total amino acids, the samples ofhydrolysate A are subjected, beforehand, to acid hydrolysis via theaction of 6N HCl for 12 hours at 110° C. under vacuum, whereas, forassaying the free amino acids, sulfosalicylic acid is added, beforehand,to the samples of hydrolysate A and the mixture is centrifuged in orderto cause the proteins present in the samples to precipitate.

Table II below shows the solids content and the contents of inorganicmaterial, of soluble proteins, of total sugars, of glycogen, of totalamino acids and of free amino acids exhibited, respectively, byhydrolysate A, the ground material from oysters and the aqueous oysterextract.

The solids contents are expressed as percentages relative to thelyophilized weight (% w/w) of the samples, except in the case of theground material, for which the solids are expressed as percentagerelative to the fresh weight (I w/w*) of the samples. The contents ofinorganic material, of soluble proteins, of total sugars, of glycogen,of total amino acids and of free amino acids are expressed aspercentages relative to the dry weights (% w/w) of the samples.

TABLE II Hydrolysate Ground Aqueous A material extract Solids 96.2310.20 95 (% w/w) (% w/w*) (% w/w) Inorganic material 36.43 37.33 37 (%w/w) Soluble proteins 13.25 30 15 (% w/w) Total sugars 8.52 6.63 3.7 (%w/w) Glycogen 1.29 1 1.5 (% w/w) Total amino acids 35.1 36.7 20.15 (%w/w) Free amino acids 17.8 7 8.10 (% w/w)

Table III below itself shows the compositions of total and free aminoacids of hydrolysate A, of the ground material from oysters and of theaqueous oyster extract. The contents of each amino acid are expressed aspercentages relative to the total weight (% w/w) of the amino acidspresent in the samples.

TABLE III HYDROLYSATE A GROUND MATERIAL AQUEOUS EXTRACT Total AA Free AATotal AA Free AA Total AA Free AA Amino acids (% w/w) (% w/w) (% w/w) (%w/w) (% w/w) (% w/w) Taurine 10.67 19.25 11.48 55.98 30.47 60.66Hydroxyproline — — — — — — Aspartic acid 10.08 2.45 10.43 4.79 11.260.72 Threonine 5.02 4.07 4.79 — 4.16 — Serine 4.67 6.58 5.02 2.28 4.462.21 Glutamic acid 13.26 8.08 13.49 8.60 11.91 11.46 Proline 5.12 2.204.85 7.59 — — Glycine 6.31 3.61 6.49 6.81 5.31 5.05 Alanine 5.64 6.434.39 3.21 5.80 8.01 Cysteine — — — — — — Valine 4.51 4.94 4.16 0.42 2.77— Methionine 2.13 2.76 2.06 — 1.53 — Isoleucine 4.02 4.38 3.36 — 2.58 —Leucine 6.18 7.63 6.32 0.65 4.66 — Tyrosine 3.39 5.04 3.27 — 1.73 2.95Phenylalanine 3.53 4.71 3.29 0.26 2.72 — Hydroxylysine — — — — — —Lysine 6.28 7.00 7.04 3.59 5.21 0.73 Histidine 2.33 2.92 2.92 1.70 1.24— Arginine 6.86 7.94 6.65 4.11 4.16 1.72

Table II shows that hydrolysate A has a content of total sugars greaterthan that found in the ground material and in the aqueous extract. Thisincrease is due to the destructuring of tissues caused by the enzymatichydrolysis, thus allowing greater solubilization of the sugars. Thedecrease in the content of soluble proteins which is observed betweenthe ground material and the hydrolysate is a consequence of thehydrolysis of the native proteins. This hydrolysis generates aconsiderable amount of free amino acids and peptides which arerelatively unreactive with the reagent used to assay the solubleproteins (BCA®). On the other hand, the content of inorganic materialsdoes not vary between the three preparations.

Moreover, it results from Table II that the content of free amino acidsin hydrolysate A is notably higher than the content of free amino acidsin the aqueous oyster extract, the latter being very close to that foundfor the ground material from oyster flesh. The increase in the amount offree amino acids present in hydrolysate A is directly linked to thebreaking of peptide bonds caused by the hydrolysis reaction.

However, in view of Table III, it appears that the proportion of freetaurine, which is known to have antioxidant activity, is lower inhydrolysate A than in the aqueous oyster extract. Specifically, taurinein free form represents 60.66% of the free amino acids in the aqueousoyster extract against only 19.25% in hydrolysate A.

EXAMPLE 5 Biological Activity of the Enzymatic Oyster Flesh HydrolysatesObtained in Accordance with the Invention

The biological activity of hydrolysates A and B prepared according toexample 3 is assessed via a series of experiments aimed at testing:

-   -   firstly, the ability of these hydrolysates to inhibit hemolysis        induced by introducing a peroxide radical generator, namely        2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), into a        suspension of hematocytes, and    -   secondly, the ability of these hydrolysates to protect low        density lipoproteins (LDLs) against copper-induced oxidation.        5.1—Inhibition of AAPH-Induced Hemolysis:        a) Protocol

5 ml of human blood are taken into an EDTA tube (which is immediatelyplaced in crushed ice) and centrifuged for 10 minutes at 1000 g and at4° C. The plasma is removed and the hematocytes are washed 3 times witha 9% NaCl solution with PBS buffer (pH 7.4). 200 μl of the hematocytecell pellet are then diluted in 9.8 μl of 9% NaCl solution or of PBSbuffer.

Firstly, the cell suspension obtained is brought into contact, for 10minutes, with the solutions (9% NaCl or PBS) of hydrolysate A or B, thevolume of which is calculated such that the final solution correspondsto 25, 50 and 100 mg/l. A sample: without hydrolysate constitutes thecontrol.

300 μl of a solution of AAPH preincubated at 37° C. are then introducedinto the hematocyte suspensions and the entire mixture is placed, withgentle stirring, in a water bath for 40 minutes.

In parallel, a sample of the hematocyte suspension (without AAPH orproduct) is placed at −80° C. for 1 hour.

The hematocyte lysis is assessed by measuring lactate dehydrogenase(LDH) activity using a Hitachi® 911 automatic machine. Each measurementis taken in duplicate.

The LDH activity determined on the samples placed at −80° C. correspondsto the total hematocyte hemolysis.

The LDH activity determined on the samples which did not containhydrolysate corresponds to the sensitivity of the hematocytes to the“free-radical stress” under the experimental conditions. Thismeasurement makes it possible, moreover, to verify that the experimentalconditions (hemolysis <100%) are suitable for the study.

For each concentration of hydrolysate, the LDH activity is compared tothe activity of the samples which do not contain any product, andexpressed as percentage activity.

b) Results:

Table IV below shows the mean of the percentages of inhibition (Ia)obtained for solutions of 25, 50 and 100 mg/l of hydrolysate A and ofhydrolysate B.

TABLE IV Concentration Ia (%) (mg/l) Hydrolysate A Hydrolysate B 25 3825 50 74 48 100 96 98

This table shows that the enzymatic oyster flesh hydrolysates obtainedin accordance with the invention exhibit a marked ability to inhibit thehemolysis induced by introducing a peroxide radical generator into asuspension of hematocytes, which means that they are capable ofneutralizing the oxidant effects of these peroxide radicals, since theinhibitory concentration 50 (IC₅₀) of hydrolysate A is between 25 and 50mg/l, whereas that of hydrolysate B comes to 50 mg/l.

By way of comparison, the inhibitory concentration 50 (IC₅₀) obtained byDussart (ibid) for an aqueous oyster extract is 275 mg/l.

5.2 Protection of LDLs Against Copper-Induced Oxidation:

a) Protocol;

The LDLs are prepared from 100 ml of plasma (blood taken on EDTA).Firstly, the VLDLs are removed by ultracentrifugation for 24 hours at40,000 g (density; 1.019). A second ultracentrifugation, for 24 hours at40,000 g (density: 1.063), enables the LDLs to be obtained. The LDLs arethen dialyzed for 24 hours at 4° C. against Tris-EDTA buffer, aliquotedand then stored at 4° C.

The LDLs (0.2 mg of protein/ml of solution), dialyzed beforehand againstPBS buffer, are incubated for 24 hours at 37° C. in the presence ofcopper (oxidant) and in the presence or absence of the products studied.

For each study, 3 determinations are therefore made in parallel:

-   -   LDL in the absence of copper (native LDL control),    -   LDL in the presence of 5 μM of copper sulfate (oxidized LDL        control),    -   LDL in the presence of 5 μM of copper sulfate and of increasing        concentrations of hydrolysates A and B.

After the oxidation has been stopped with BHT/EDTA, the LDL solution isdialyzed for 24 hours at +4° C. and filtered through a 0.2 μm“millipore” membrane.

The inhibitory effect of the hydrolysates with respect to the LDLoxidation by the copper is quantified by assaying 2 lipoperoxidationmarkers:

-   -   MDA (malondialdehyde), for calculating the percentage of        inhibition Ib,    -   hydroperoxides, for calculating the percentage of inhibition Ic.        MDA Assay

MDA forms, with thiobarbituric acid, when hot and in acid medium, afluorescent chromogenic complex. After extraction with normal butanol,the intensity of the fluorescence is measured using aspectrofluorometer. The MDA concentrations are determined by means of anMDA range extending from 0.2 to 1 nmol.

Hydroperoxide Assay

Hydroperoxides release iodine from a stabilized solution of potassiumiodide. The released iodine is measured by determining the opticaldensity (OD) at 365 nm.

The iodine concentration of the sample is then calculated from theextinction coefficient ε (=2.46 104, 1 cm, 1M) of this element.

b) Results:

Tables V and VI below show, respectively, the percentages of inhibition(Ib) and (Ic) as obtained for solutions of 25, 50, 100 and 250 mg/l ofhydrolysate A and of hydrolysate B.

TABLE V Concentration Ib (%) (mg/l) Hydrolysate A Hydrolysate B 25 −9 4050 75 82 100 73 86 250 86 89

TABLE VI Concentration Ic (%) (mg/l) Hydrolysate A Hydrolysate B 25 1071 50 100 100 100 100 100 250 100 100

These tables show that the enzymatic oyster flesh hydrolysates obtainedin accordance with the invention also have a pronounced ability tooppose copper-induced LDL oxidation, this being an ability which may belinked to a chelating effect with respect to metals.

1. A method of preparing a free-radical scavenging composition,comprising hydrolyzing oyster flesh using a protease to obtain anenzymatic oyster hydrolysate, wherein the hydrolysate is obtained usinga method comprising the following steps: a) grinding predrained oysterflesh, b) diluting the ground material in water, at a groundmaterial/water ratio of between 30/70 and 70/30 (m/v), c) hydrolyzingthe ground material thus diluted with subtilisin at a pH ofapproximately, 8 and at a temperature of approximately 60° C. for aperiod of time sufficient for the hydrolysate to exhibit a degree ofprotein hydrolysis at least equal to 50%, d) stopping the hydrolysis byinactivation of the subtilisin, and e) collecting the liquid phase ofthe hydrolysate.
 2. A method of preparing a fee-radical scavengingcomposition, comprising hydrolyzing oyster flesh using a protease toobtain an enzymatic oyster hydrolysate, wherein the hydrolysate isobtained using a method comprising the following steps: a) grindingpredrained oyster flesh, b) diluting the ground material in water, at aground material/water ratio of between 30/70 and 70/30 (m/v), c)hydrolyzing the ground material thus diluted with pepsin, at a pH ofapproximately 2 and at a temperature of approximately 40° C. for aperiod of time sufficient for the hydrolysate to exhibit a degree ofprotein hydrolysis at least equal to 50%, d) stopping the hydrolysis byinactivation of the pepsin, and e) collecting the liquid phase of thehydrolysate.
 3. A method of preparing a free-radical scavengingcomposition, comprising hydrolyzing oyster flesh using a protease toobtain enzymatic oyster hydrolysate, wherein the hydrolysate is obtainedusing a method comprising the following steps: a) grinding predrainedoyster flesh, b) diluting the ground material in water, at a groundmaterial/water ratio of between 30/70 and 70/30 (m/w), c) hydrolyzingthe ground material thus diluted with trypsin, at a pH of approximately8 and at a temperature of approximately 37° C., for a period of timesufficient for the hydrolysate to exhibit a degree of protein hydrolysisat least equal to 50%, d) stopping the hydrolysis by inactivation of thetrypsin, and e) collecting the liquid phase of the hydrolysate.
 4. Afood supplement comprising a free-radical scavenging compositionobtained by the method of claim
 1. 5. A food supplement comprising afree-radical scavenging composition obtained by the method of claim 2.6. A food supplement comprising a free-radical scavenging compositionobtained by the method of claim 3.